Biological Polymers: A Focus on Dragline Spider Silk

1
Biological Polymers A Focus on Dragline Spider
Silk Spidroin Protein
SEM of an ion etched silk fibroin fiber
Picture courtesy of Chang et. Al., Polymer 46
7909 (2005)
2
Biological Polymers

• 3 Main classes of biological polymers
• Nucleic acid polymers
• Linear informational polymers comprised of 4
  nucleotide monomers
• Polysaccharides
• Branching storage/structural polymers comprised
  of one of a few select monosaccharide monomers
• Proteins and peptides
• Linear informational polymers comprised of 20
  standard amino acid monomers
• Nucleic acids and proteins are considered
  informational because the sequence of monomers in
  the polymers is
• Nonrandom
• Significant to function
• A similar argument can be made for branched
  carbohydrates comprised of different monomers.

• General Functions of Biological Polymers
• Nucleic Acids
• Information storage (genome)
• Translational molecules (mRNA tRNA)
• Biological catalysts (RNA ribozymes)
• Carbohydrates
• Energy storage (glycogen)
• Structural (cellulose cell walls or chitin
  exoskeletons)
• Recognition (carbohydrates of glycoproteins and
  glycolipids)
• Proteins
• Structural (fibrous proteins)
• Biological catalysts (enzymes)
• Recognition (immunoglobulins)

3
Biopolymer Synthesis Via Condensation

• Implies that monomers must have hydrogen-bearing
  and hydroxyl moieties.
• Directed polymerization is accomplished by
  chemically activating monomers via
• Direct activation using ATP or Coenzyme A
• The use of a carrier molecule (i.e. tRNA)
• Polymerization dictates that biological polymers
  have chemically distinct ends.

Scheme of biopolymer macromolecular assembly
Figure 2-17 Becker et. Al., World of the Cell
6th Ed.
4
Biopolymers Utilize a Variety of Functional
Groups for Polymerization by Condensation
Nucleic acid structure highlighting chemically
distinct ends
Common functional groups employed for biopolymer
formation

• Candidate functional groups for condensation
  polymerization must either act as a nucleophile
  or electrophile

Left Figure 1-2 Voet et. Al., Fundamentals of
Biochemistry. Right Figure 3-6a Voet et. Al.,
Fundamentals of Biochemistry.
5
Efficiency of Biopolymer Synthesis
Scheme of amino acid polymerization by
condensation

• Biopolymer condensation is spontaneous and
  relatively rapid at moderate temperatures in
  aqueous environments.
• Chemical initiators are not required.
• The use of biological catalysts (enzymes) and
  activating molecules
• Improves efficiency to favor polymerization over
  depolymerization (hydrolysis) by moving the
  reaction away from equilibrium
• Makes biopolymer formation kinetically competent
  to support life

Scheme of ribosomal (catalytic) protein synthesis
Top Figure 4-3 Voet et. Al., Fundamentals of
Biochemistry. Right Figure 26-28 Voet et. Al.,
Fundamentals of Biochemistry.
6
3-D Structure is Intimately Related to Function

• Three-dimensional arrangements of biological
  polymers are more important for function than the
  chemical nature and composition of the monomers.
• Examples
• The tertiary structure of proteins is largely
  responsible for biological activity.
• The double helical structure of DNA is
  responsible for stability, replication
  efficiency, and packing in small cellular
  volumes.
• The 3-D arrays of complex carbohydrates
  determines optimal intracellular storage
  conditions and recognition properties.

7
The Hierarchal Structure of Proteins

• Primary Structure
• Amino acid sequence from N- to C-terminus
• Ultimately determines all higher order structure
  and function
• Driven and stabilized by covalent bonds
• Secondary Structure
• Local, spatial interactions between functional
  groups of the protein backbone
• Driven and stabilized by the hydrogen bond
• Not usually a determinant of function
• Tertiary Structure
• Three-dimensional folding of a polypeptide
• Driven and stabilized largely by weak,
  hydrophobic interactions
• Often dictates biological activity
• Quaternary Structure
• Specific interactions between two or more
  proteins
• Can be driven and stabilized by any combination
  of bond types

Figure illustrating the four hierarchal levels of
protein structure
Figure 3-6 Becker et. Al., World of the Cell 6th
Ed.
Structure is a consequence of sequence. Function
is a consequence of structure.
8
Spider Dragline Silk

• Spiders have 7 different gland-spinneret
  complexes
• Each synthesizes a unique blend of structural
  polymer as a fiber with unique properties
• Multiple fibers can be spun simultaneously
• Dragline silk is used by spiders to build the
  frame and radii of their nets and as lifelines.
• Dragline silk is produced by the largest gland
  (major ampullate) and is believed to have the
  most desirable properties for commercial use.
• Potential applications include
• Biomedical sutures
• Scaffolds for tissue engineering (bone
  ligament)
• Body armor

Photograph illustrating spider silk formation
stress-strain curves for dragline and viscid
spider silk
Top Picture courtesy of Tiller et. Al.,
1996. Bottom Figure courtesy of Gosline et. Al.,
1999.
9
Macromolecular Structure of Silk Spidroin
SEM of untreated and toluene treated spidroin
fibers

• Major ampullate dragline silk is comprised of two
  proteins joined together via 3 5 disulfide
  bonds near their C-termini
• Spidroin 1
• Spidroin 2
• The average diameter of major ampullate dragline
  silk spidroin ? 2.53 0.4 ?m.
• Mucopolysaccharide is infused within, and on the
  surface of the silk fibers (removed by toluene
  treatment).

Figure courtesy of Rengasamy et. Al., 2005.
10
Primary Sequence of Spider Silk Spidroin

• Four motifs exist in the primary structure
• GPGXX (X often Q)
• An or (GA)n
• GGX
• Spacer regions

• Two residues predominate in the primary sequence
• 42 Glycine
• 25 Alanine
• Glu, Gln, Ser and Tyr are also prominent
• Cys is concentrated near the C-terminus

Sequences of major ampullate spidroin highlighting
motif transitions
Figure courtesy of Gosline et. Al., 1999.
11
Secondary Structure Predictions from the Primary
Sequence
DOQSY Spectra and Ramachandran diagrams of silk
spidroin fibers

• Double-quantum single-quantum correlation for
  static sample (DOQSY) NMR can measure the
  relative orientation of the peptide backbone
  carbonyl orientation when if 13C is present.
• Feeding deuterated and 13C-L-alanine to spiders
  reveals that 40 of total alanine is involved in
  crystalline protein structure.
• Chou-Fasman prediction of spidroin 2? structure
  indicates the ?-helix and turns should
  predominate.
• Ala P? 1.42, P? 0.83, Pturn 0.66
• Gly P? 0.57, P? 0.75, Pturn 1.56
• Glu P? 1.51, P? 0.37, Pturn 0.74
• Gln P? 1.11, P? 1.10, Pturn 0.98
• Ser P? 0.77, P? 0.75, Pturn 1.43
• Tyr P? 0.69, P? 1.47, Pturn 1.14
• Cys P? 0.70, P? 1.19, Pturn 1.19

Figure courtesy of van Beek et. Al., 2002.
Alanine torsion angles indicate ?? 135?, ??
150? What does this data suggest?
12
Circular Dichroism Spectra Indicates ?-Sheet
Structure
CD Spectra and cooperative thermal transitions of
spidroin segments against an ?-helical background

• Circular dichroism measures the optical activity
  of proteins in the far UV-region.
• Dissymmetry due to bias towards L-amino acids and
  the preferential twists of secondary structure
  can be distinguished.
• ?-helices have a strong positive band at 192 nm
  and two negative bands at 208 and 222 nm.
• CD spectra reveal no ?-helices and a cooperative
  and reversible disruption of protein 2?
  structure.
• Fourier transform infrared spectroscopy (FTIR)
  confirms that ?-sheets are oriented parallel to
  the fiber axis.

Figure courtesy of Huemmerich et. Al., 2004.
13
X-Ray Crystallography Reveals A Composite,
Hierarchal Block Co-Polymer
Summary figure of spidroin crystal structure in
supercontracted vs. fibers

• Poly-Ala or (GA)n stretches form ?-sheets.
• Glu and Tyr limit the size and spacing of
  ?-sheets by forcing loops to form and interact
  with the surrounding matrix.
• ?-sheets stack on top of one another with crystal
  dimensions of ? 2nm X 5 nm X 7 nm.
• ?-sheet crystals form intermolecular connections
  and are large and abundant enough to act as
  reinforcing filler particles to stiffen and
  strengthen the overall structure.
• Major ampullate silk structure can be summarized
  as a crystal cross-linked, crystal-reinforced
  polymer network.

Figure courtesy of Gosline et. Al., 1999.
14
Physicochemical Analysis of Major Ampullate
Spidroin
Differential scanning calorimetry thermal
mechanical analysis of spidroin fibers

• Differential scanning calorimetry shows a broad
  endotherm with a peak at ? 9095 ?C, consistent
  with the loss of water, and is stable up to ?250
  ?C.
• Thermogravimetric analysis shows a two-step
  degradation profile above 150 ?C
• First step in the range of 200501 ?C corresponds
  to the destruction of the amino acid side chains
• Second step in the range of 501896 ?C
  corresponds to destruction of the peptide bonds
• Thermal mechanical analysis shows a change in the
  thermal expansion coefficient (?) from 6.59 X
  104 to 8.2 X 103 at 186.4 ?C (low glass
  transition temperature).

Figures courtesy of Rengasamy et. Al., 2005.
15
Physical Parameters of Major Ampullate Spidroin

• Stress (?) the normalized force (F) such that
• ? F/A (A initial cross-sectional area of the
  fiber)
• Strain (?) the normalized deformation such
  that
• ? ??L/L0 (L0 initial fiber length and ?L
  change in fiber length)
• A stress-strain curve (? vs. ?) gives
• Stiffness of the material (slope)
• Strength of the material (?max) as the maximum
  value of stress at the time the material fails
• Extensibility of the material (?max) as the
  maximum value of strain at the time the material
  fails
• The integrated area under the stress-strain curve
  gives the energy required to break the material
  and is a quantification of toughness

16
Stress-Strain Curves in Different Solvents
Reveals Unique Properties

• Silk shrinks by 40 50 and softens/weakens as a
  function of solvent EtOH lt MetOH lt Water lt Urea
• The transition supercontraction is a function of
  solvent dielectric
• Big problem for engineering
• Beneficial for the spider in environmental
  adaptation
• Water and methanol act as plasticizers, and
  insinuates itself into the spidroin polymer to
  reduce inter-fiber interactions
• Decreases the elastic modulus
• Decreases strength and toughness
• Solvent absorbed during supercontraction is
  associated only with amorphous (non-crystalline)
  regions of the spidroin structure.

Stress-strain curves of major ampullate spidroin
in different solvents
Figures courtesy of Shao et. Al., 1999.
17
Dried Spidroin Fibers Do Not Recover Fully
Stress-strain curves of major ampullate spidroin
in before and after submersion drying in
different solvents

• Silk submerged in high dielectric solvents
• Exhibits a stress-strain profile more consistent
  with commercial rubber
• Submerged silk that is dried only partially
  recovers
• Forms a semi-crystalline polymer
• Stiffness decreases by 3 orders of magnitude
• Mucopolysaccharide infusion and coating may
  partially protect spidroin from supercontraction.

Figures courtesy of Shao et. Al., 1999.
18
Multiple Loading-Unloading Decreases Toughness
and Extensibility Only Marginally After Drying
Successive stress-strain curves of major
ampullate spidroin after submersion drying in
water

• Elastic recovery after submersion drying is
  between 80 90 of maximum after stretching to
  70 of breaking elongation.

Figures courtesy of Shao et. Al., 1999.
19
High-Strain-Rate Impact Reveals Hysteresis
High-strain-rate analysis approximating common
loads experienced by spidroin fibers

• When dragline silk is first under strain it
  absorbs energy as the molecular chains reorient
  and slip against each other as H-bonds break.
• After stretching, chains settle into a stable
  conformation.
• Friction between chains and reformation of
  H-bonds induce a permanent set to prevent full
  recovery at relaxation.
• A hysteresis value of 65
• Allows 65 of transmitted kinetic energy to be
  absorbed and transformed into heat
• Prevents prey from catapulting out of the web
• Represents a balance between strength and
  extensibility yielding enormous toughness

Figures courtesy of Gosline et. Al., 1999.
20
Stress-Strain Comparisons With High-Performance
Polymers
Table courtesy of Gosline et. Al., 1999.

• Major ampullate spidroin is amongst the stiffest
  and strongest biomaterials known.
• Large extensibility (stretch), in spite of
  decreased strength, makes silk tougher than
  engineering materials.
• Major ampullate spidroin has hard elastic
  properties that can outperform all synthetic
  fibers when energy absorption is important.
• A Kevlar fiber of exactly the same breaking
  tension, but with an ?max one order of magnitude
  lower than spidroin would support a load less
  than 40 of a comparable silk fiber.
• Major ampullate silk spidroin is 5-times stronger
  than steel by weight.

21
Rationalizing Spidroin Properties With Fiber
Structure
Proposed model for dragline silk fiber

• GPGXX (GPGQQ)
• Likely a ?-turn spiral
• Contributes to elasticity and connects
  crystalline sheets
• P allows for retraction after stretching by
  providing torque
• Serves as a focal point for retractive forces
  after stretching
• (GA)n / An
• Crystalline ?-sheets that provide high tensile
  strength
• Form zipper-like stacking of interdigitating
  sheets
• GGX
• 310 helix
• Likely important for fiber alignment
• Spacers
• Contributes to both elasticity and
  supercontraction
• Serves as the matrix for embedding the
  crystalline regions of the polymer
• May prevent premature fiber formation in the
  spider gland

Figure courtesy of van Beek et. Al., 2002.
22
Biology of the Major Ampullate Gland

• Silk proteins are stored in a liquid crystal form
  (elongated flexible rods) while in the gland.
• Fibers are not formed until the protein passes
  trough the duct leading to the spinneret.
• During thread assembly and spinning
• Water, sodium and chloride are removed
• Lyotropic ions (K and PO43) induce liquid
  crystal formation by increasing the surface
  tension of water and increasing hydrophobic
  interactions by changing structural water to bulk
  water
• pH drops from 6.9 to 6.3
• The mechanical stress of funneling through the
  gland and passing through the spinneret induced
  fiber alignment and assembly of the fiber by
  extensional flow
• Fibers must be dehydrated to initiate ?-sheet
  formation and crystallization.

Micrograph of a single spider spinneret
highlighting internal anatomy
Image courtesy of www.hubcap.clemson,edu/ellisom
/ biomimeticmaterials/files/spinningsystems.htm.
23
Considerations for Engineered Dragline Silk

• Expression of authentic spider silk in bacterial
  hosts is inefficient since some eukaryotic codons
  are not translated efficiently in bacteria.
• Gene manipulation and amplification by PCR is
  difficult due to the repetitive nature of silk.
• Drink your goat-milk silk!!!!
• Dehydration and extensional flow must be
  reproduced in vitro to produce silk with the
  expected high strength, extensibility and
  toughness of native dragline silk.

24
Preliminary Attempts at Engineering Dragline Silk
Has Been Successful

• Artificial spinning procedures of engineered
  dragline silk in hexafluoroisopropanol have
  produced films with a tensile strength on the
  order of 10 GPa and an elongation/extensibility
  3-fold higher than native dragline silk.
• Alteration of spinning conditions can markedly
  improve select characteristics of engineered
  silk
• Faster spinning produces stronger, more brittle
  fibers
• Slower spinning produces weaker, more elastic
  fibers
• The major hurdle for mass production and
  commercial application is producing engineered
  silk in mass quantity.

25

• Drink your goat milk!!!!
• Questions, Comments, Screams of Fury and Pain???

26
References (Alphabetical)

• Allcock Lampe. Contemporary Polymer Chemistry
  2nd Ed. Prentice Hall, Inc., 1990.
• Altman et. Al. Biomaterials 24 401416, 2003.
• Becker et. Al. The World of the Cell 6th Ed.
  Pearson/Benjamin Cummings Press, 2005.
• Chang et. Al. Polymer 46 79097917, 2005.
• Gosline et. Al. J. Exp. Biol. 202 32953303,
  1999.
• Hinman et. Al. TIBTECH 18 374379, 2000.
• Huemmerich et. Al. Biochemistry 43 1360413612,
  2004.
• Rengasamy et. Al. AUTEX Res. J. 5 3039, 2005.
• Rising et. Al. Zoo. Sci. 22 273281, 2005.
• Shao, Z. Vollrath, F. Polymer 40 17991806,
  1999.
• Tirrell, D. Science 271 39 40, 1996.
• www.hubcap.clemson,edu/ellisom/biomimeticmaterial
  s/files/spinningsystems.htm.
• van Beek et. Al. PNAS 99 1026610271, 2002.
• Voet et. Al. Fundamentals of Biochemistry. John
  Wiley Sons, Inc., 2001.